Novel C-17-Heteroaryl Steroidal Cyp17 Inhibitors/Antiandrogens, In Vitro Biological Activities, Pharmacokinetics and Antitumor Activity

ABSTRACT

Described are steroidal C-17 benzoazoles, pyrimidinoazoles (azabenzoazoles) and diazines. Methods for their synthesis are also described, which include methods having a step of nucleophilic vinylic “addition-elimination” substitution reaction of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene or analogs thereof and benzoazole or pyrimidinoazole nucleophiles and methods having a palladium catalyzed cross-coupling reaction of 17-iodoandrosta-5,16-dien-3β-ol or analogs thereof with tributylstannyl diazines. The compounds are potent inhibitors of human CYP 17 enzyme as well as potent antagonists of both wild type and mutant androgen receptors (AR). The compounds are useful for the treatment of human prostate cancer.

The present application is a divisional application of and claims thebenefit of U.S. application Ser. No. 11/817,550, filed on Mar. 14, 2008,which claims the benefit and filing date of U.S. Provisional ApplicationSer. No. 60/657,390, filed on Mar. 2, 2005, each of which isincorporated by reference herein in its entirety.

This invention was made with government support under grant No. CA27440awarded by the National Institutes of Health (NIH). The government hascertain rights in the invention.

The present invention provides new chemical entities, particularlysteroidal C-17 benzoazoles, pyrimidinoazoles (azabenzoazoles) anddiazines. It is also provides methods for the synthesis of thebenzoazoles, pyrimidinoazoles and diazines. In one embodiment, themethods for synthesizing benzoazoles or pyrimidinoazoles comprise a stepof nucleophilic vinylic “addition-elimination” substitution reaction of3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene or analogs thereof andbenzoazole or pyrimidinoazole nucleophiles. In another embodiment, themethods for synthesizing diazines comprise a palladium catalyzedcross-coupling reaction of 17-iodoandrosta-5,16-dien-3β-ol or analogsthereof with tributylstannyl diazines.

Compounds of the present invention are potent inhibitors of human CYP17enzyme as well as potent antagonists of both wild type and mutantandrogen receptors (AR). The most potent CYP17 inhibitors were:3β-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (5, code namedVN/124-1), 3β-hydroxy-17-(5¹-pyrimidyl)androsta-5,16-diene (15) and17-(1H-benzimidazole-1-yl)androsta-4,16-diene-3-one (6), with IC₅₀values of 300, 500 and 915 nM, respectively. Compounds 5, 6, 14 and 15were effective at preventing binding of ³H-R1881 (methyltrienolone, astable synthetic androgen) to both the mutant and LNCaP AR and thewild-type AR, but with a 2.2 to 5-fold higher binding efficiency to thelatter. Compounds 5 and 6 were also shown to be potent pure ARantagonists. The cell growth studies showed that 5 and 6 inhibit thegrowth of DHT-stimulated LNCaP and LAPC4 prostate cancer cells with IC₅₀values in the low micromolar range (i.e., <10 μM). Their inhibitorypotencies were comparable to that of casodex but remarkably superior tothat of flutamide. The pharmacokinetics of compounds 5 and 6 in mice wasinvestigated. Following s.c. administration of 50 mg/kg of 5 and 6, peakplasma levels of 16.82 and 5.15 ng/mL, respectively occurred after 30 to60 min, both compounds were cleared rapidly from plasma (terminalhalf-lives of 44.17 and 39.93 min, respectively) and neither wasdetectable at 8 h. Remarkably, compound 5 was rapidly converted into ametabolite tentatively identified as17-(1H-benzimidazol-1-yl)androsta-3-one. When tested in vivo, 5 provedto be very effective at inhibiting the growth of androgen-dependentLAPC4 human prostate tumor xenograft, while 6 was ineffective. Compound5 (50 mg/kg/twice daily) resulted in a 93.8% reduction (P=0.00065) inthe mean final tumor volume compared with controls, and it was alsosignificantly more effective than castration. To our knowledge, this isthe first example of an anti-hormonal agent (an inhibitor of androgensynthesis (CYP17 inhibitor)/antiandrogen) that is significantly moreeffective than castration in suppression of androgen-dependent prostatetumor growth. In view of these impressive anti-cancer properties,compound 5 and others can be used for the treatment of human prostatecancer. Prostate cancer (PCA) is the most common malignancy andage-related cause of cancer death worldwide. Apart from lung cancer, PCAis the most common form of cancer in men and the second leading cause ofdeath in American men. In the United States this year (2004), anestimated 230,000 new case of prostate cancer will be diagnosed andabout 23,000 men will die of this disease (Jemal et al., CancerStatistics, 2004. CA Cancer J. Clin., 2004, 54, 8-29). During the periodof 1992 to 1999, the average annual incidence of PCA among AfricanAmerican men was 59% higher than among Caucasian men, and the averageannual death rate was more than twice that of Caucasian men (AmericanCancer Society—Cancer Facts and Figures 2003). Androgens play animportant role in the development, growth, and progress sion of PCA(McConnell, J. D., “Physiological basis of endocrine therapy forprostatic cancer”, Urol. Clin. North Am., 1991, 18: 1-13). The two mostimportant androgens in this regard are testosterone (T) anddihydrotestosterone (DHT). The testes synthesize about 90% of T and therest (10%) is synthesized by the adrenal glands. T is further convertedto the more potent androgen DHT by the enzyme steroid 5α-reductase thatis localized primarily in the prostate (Bruchovsky et al., “Theconversion of testosterone to 5α-androstan-17β-ol-3-one by rat prostatein vivo and in vitro”, J. Biol. Chem., 1968, 243, 2012-2021). Huggins etal. introduced androgen deprivation as therapy for advanced andmetastatic PCA in 1941 (Huggins et al. “Studies on prostatic cancer: 2.The effects of castration on advanced carcinoma of the prostate gland.”,Arch. Surg., 1941, 43, 209-212). Thereafter, androgen ablation therapyhas been shown to produce the most beneficial responses in multiplesettings in PCA patients (Denmeade et al. “A history of prostate cancertreatment.” Nature Rev. Cancer, 2002, 2: 389-396). Orchidectomy (eithersurgical or medical with a GnRH agonist) remains the standard treatmentoption for most prostate cancer patients. Medical and surgicalorchidectomy reduces or eliminates androgen production by the testes butdoes not affect androgen synthesis in the adrenal glands. Severalstudies have reported that a combination therapy of orchidectomy withantiandrogens, to inhibit the action of adrenal androgens, significantlyprolongs the survival of PCA patients (Crawford, et al., “A controlledtrial of leuprolide with and without flutamide in protatic carcinoma”,N. Engl. J. Med., 1989, 321, 419-424; Crawford, et al., “Treatment ofnewly diagnosed state D2 prostate cancer with leuprolide and flutamideor leuprolide alone, Phase III: intergroup study 0036”, J. Urol., 1992,147: 417A; and Denis, L., “Role of maximal androgen blockade in advancedprostate cancer”, Prostate, 1994, 5 (Suppl.), 17s-22s). In a recentfeatured article by Mohler and colleagues (Mohler et al., “The androgenaxis in recurrent prostate cancer”, Clin. Cancer Res., 2004, 10,440-448) it was clearly demonstrated that T and DHT occur in recurrentPCA tissues at levels sufficient to activate androgen receptor. Inaddition, using microarray-based profiling of isogenic PCA xenograftmodels, Sawyer and colleagues (Chen et al., “Molecular determinants ofresistance to antiandrogen therapy.” Nat. Med., 2004, 10, 33-39) foundthat a modest increase in androgen receptor mRNA was the only changeconsistently associated with the development of resistance toantiandrogen therapy. Potent and specific compounds that inhibitandrogen synthesis in the testes, adrenals, and other tissue may be moreeffective for the treatment of PCA (Njar, V. C. O.; Brodie, A. M. H.,“Inhibitors of 17α-hydroxylase-C₁₇₋₂₀-lyase (CYP17): Potential agentsfor the treatment of prostate cancer”, Current Pharm. Design, 1999, 5:163-180).

In the testes and adrenal glands, the last step in the biosynthesis of Tinvolves two key reactions, which act sequentially and they are bothcatalyzed by a single enzyme, the cytochrome P450 monooxygenase17α-hydroxylase/17,20-lyase (CYP17) (Hall, P. F., “Cytochrome P-450C_(21scc): one enzyme with two actions: Hydroxylase and lyase”, J.Steroid Biochem. Molec. Biol., 1991, 40, 527-532). Ketoconazole, as anantifungal agent and by virtue of inhibiting P450 enzymes, is also amodest CYP17 inhibitor and has been used clinically for the treatment ofPCA (Trachtenberg et al., “Ketoconazole: A novel and rapid treatment foradvanced prostatic cancer”, J. Urol. 1983, 130, 152-153). It is reportedthat careful scheduling of treatment can produce prolonged responses inotherwise hormone-refractory prostate cancer patients (Muscato et al.,“Optimal dosing of ketoconazole and hydrocrtisone leads to longresponses in hormone refractory prostate cancer”, Proc. Am. Assoc.Cancer Res., 1994, 13: 22 (Abstract)). Furthermore, ketoconazole wasfound to retain activity in advanced PCA patients with progressiondespite flutamide withdrawal (Small et al., “Ketoconazole retainsactivity in advanced prostate cancer patients with progression despiteflutamide withdrawal”, J. Urol., 1997, 157, 1204-1207). Although,ketoconazole has now been withdrawn from use because of liver toxicityand other side effects this suggests that more potent and selectiveinhibitors of CYP17 could provide useful agents for treating thisdisease, even in advanced stages and in some patients who may appear tobe hormone refractory.

A variety of potent steroidal and non-steroidal inhibitors of CYP17 havebeen reported and some have been shown to be potent inhibitors oftestosterone production in rodent models (Njar and Brodie, above).Recently, Jarman and colleagues have described the hormonal impact oftheir most potent CYP17 inhibitor, abiraterone in patients with prostatecancer (O'Donnell et al., “Hormonal impact of the17α-hydroxylase/C17,20-lyase inhibitors abiraterone acetate (CB7630) inpatients with prostate cancer”, Br. J. Cancer, 2004, 90: 2317-2325).Some of our potent CYP17 inhibitors have been shown to also inhibit5α-reductase and/or are potent antiandrogens with potent antitumoractivity (Njar and Brodie, above, and Long et al., “Antiandrogeniceffects of novel androgen synthesis inhibitors on hormone-dependentprostate cancer.” Cancer Res., 2000, 60, 6630-6640). C₁₇₋₂₀-lyasecatalyzes both the 17α-hydroxylation and the cleavage of the C₁₇₋₂₀-sidechain during the conversion of the 21-carbon steroids pregnenolone andprogesterone to the 19-carbon androgens dehydroepiandrosterone andandrostenedione, respectively. Further illustrative of the background ofthe invention are U.S. Pat. Nos. 5,994,335; 6,200,965; and, 6,444,683.

We have discovered a series of potent CYP17 inhibitors/antiandrogens,the 17-benzoazoles, 17-pyrimidinoazoles and 17-diazines (see, e.g.,Schemes 1 and 2, for examples of preparation of compounds which can beanalogously applied to other structures, as described below). Thestimulus for preparing these C-17 heteroaryl steroids was based on ourdesire to incorporate benzimidazole, benzotriazole, pyrimidinoazole anddiazine moieties, so-called “privileged substructures” (Nicolaou et al.,“Natural product-like combinatorial libraries based on privilegedstructures. 1. General principles and solid-phase synthesis ofbenzopyrans”, J. Am. Chem. Soc., 2000, 122, 9939-9953. “Privilegedstructures”, a term originally introduced by Evans et al. (J. Med.Chem., 1988, 31, 2235-2246) to describe structural motifs capable ofinteracting with a variety of unrelated molecular targets) in the newmolecules. These scaffolds, especially the benzimidazole scaffold,continue to receive extensive attention in medicinal chemistry becauseof their diverse portfolio of biological activities and also as entitiesof a variety of useful drugs (Nicolaou et al., above).

The C-17 heteroaryl steroid compounds of the invention are of thefollowing general formula I:

wherein:

-   -   the ABC ring structure is the A, B and C ring portions of a        steroid or analog thereof, which are optionally substituted;    -   the        bond at the 16,17 position is a double bond or, when the        compound is 17-(1H-benzimidiazol-1-yl)androst-3-one, a single        bond; and    -   X is an optionally substituted benzimidazole, benzotriazole,        pyrimidinoimidazole (purine), pyrimidinotriazole or diazine; the        benzimidazole, benzotriazole, and pyrimidinoimidazole groups        being bonded to the steroid residue through a nitrogen atom on        the 5-membered ring; and, the diazine groups being bonded to the        steroid residue through a carbon atom on the diazine ring.

Pharmaceutically acceptable salts of these compounds are also includedin the invention.

The optional substitution for the ABC ring structure includes one ormore of: alkyl and halogenated alkyl (preferably C₁₋₆); alkenyl andhalogenated alkenyl (preferably C₁₋₆) including where the double bond isdirectly attached to the ring structure; halogen; amino; aminoalkylene;hydroxyimino; and hydroxy. Further optionally, hydrogen substituents onadjacent carbon atoms of the ABC ring structure may be removed andreplaced by an additional bond between the adjacent carbon atoms toresult in a double bond between these carbons in the ring structure.Preferred optional substitutions on the ABC ring structure are methylgroups at the 10 and/or 13 positions of the ring structure.

The optional substitution for the benzimidazole, benzotriazole,pyrimidinoimidazole, pyrimidinotriazole or diazine structures includehalogen, amino, aminoalkylene, hydroxy, —SH, —S-alkyl, alkyl andhalogenated alkyl (preferably C₁₋₆). These optional substituents will beon ring carbon atoms of the benzimidazole, benzotriazole,pyrimidinoimidazole, pyrimidinotriazole or diazine structures.

The benzimidazole, benzotriazole, pyrimidinoimidazole,pyrimidinotriazole or diazine structures are of the following formulae,respectively:

wherein the * indicates the point of attachment to the steroid residue.

In one preferred embodiment, the ABC ring structure has a C ring whichhas no substitution except for preferably alkyl, particularly methyl,substitution at the carbon shared with the D ring which is adjacent theattachment to the C-17 heteroaryl substitution, i.e., the 13-position.

In another preferred embodiment, the A, B and C rings of the ABC ringstructure have a conventional structure based on3β-hydroxy-androsta-5,16-diene or 3-oxo-androsta-5,16-diene. But inanother embodiment the A and B rings have one of the followingstructures 1-25:

The following lists the chemical names of compounds having the AB ringsas in 1-25, and the C and D rings conventional, wherein X isbenzamidazole. Analogous compounds wherein X is benzotriazole,pyrimidinoimidazole, pyrimidinotriazole, pyrazine or pyrimidine are alsocontemplated.

-   Compound 1:    3β-Hydroxy-3α-methyl-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene-   Compound 2: 3β-Fluoro-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene-   Compound 3: 3β-Chloro-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene-   Compound 4: 3β-Bromo-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene-   Compound 5: 3β-Iodo-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene-   Compound 6: 3β-Amino-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene-   Compound 7: 17-(1H-benzimidazol-1-yl)-androsta-3,5,16-triene-   Compound 8: 17-(1H-benzimidazol-1-yl)-androsta-2,4,16-triene-   Compound 9:    17-(1H-benzimidazol-1-yl)-3-methyleneandrosta-5,16-triene-   Compound 10:    17-(1H-benzimidazol-1-yl)-3-methyleneandrosta-4,16-triene-   Compound 11:    3,3-Difluoro-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene-   Compound 12:    3,3-Difluoro-17-(1H-benzimidazol-1-yl)-androsta-4,16-diene-   Compound 13:    17-(1H-benzimidazol-1-yl)-3-methyleneandrosta-2,4,16-triene-   Compound 14:    17-(1H-benzimidazol-1-yl)-3-methyleneandrosta-2,4,6,16-tetraene-   Compound 15:    3,3-Difluoro-17-(1H-benzimidazol-1-yl)-androsta-2,4,16-triene-   Compound 16:    3,3-Difluoro-17-(1H-benzimidazol-1-yl)-androsta-2,4,6,16-tetraene-   Compound 17:    3-Hydroxyimino-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene-   Compound 18:    3-Hydroxyimino-17-(1H-benzimidazol-1-yl)-androsta-4,16-diene-   Compound 19:    3-Hydroxyimino-17-(1H-benzimidazol-1-yl)-androsta-2,4,16-triene-   Compound 20:    3-Hydroxyimino-17-(1H-benzimidazol-1-yl)-androsta-2,4.6,16-diene-   Compound 21:    3-Hydroxy-17-(1H-benzimidazol-1-yl)-estra-1,3,5(10),16-tetraene-   Compound 22:    3-Fluoro-17-(1H-benzimidazol-1-yl)-estra-1,3,5(10),16-tetraene-   Compound 23:    3-Chloro-17-(1H-benzimidazol-1-yl)-estra-1,3,5(10),16-tetraene-   Compound 24:    3-Bromo-17-(1H-benzimidazol-1-yl)-estra-1,3,5(10),16-tetraene-   Compound 25:    3-Iodo-17-(1H-benzimidazol-1-yl)-estra-1,3,5(10),16-tetraene

Examples of optional substituents for the heteroaryl ring, X, are shownby the following structures 26-40 wherein X is benzimidazole. Analogouscompounds wherein X is substituted benzotriazole, pyrimidinoimidazole,pyrimidinotriazole, pyrazine or pyrimidine are also contemplated.

Other examples of optional substituents for the heteroaryl ring, X, areshown by the following structures 41-46 wherein X is substitutedC-17-azabenzimidazole (i.e., pyrimidinoimidazole or purine). Analogouscompounds wherein X is substituted benzimidazole, benzotriazole,pyrimidinotriazole, pyrazine or pyrimidine are also contemplated.

Particularly preferred compounds are those of the following structuresM5, M6, M9 and M10.

The inhibitory activities of these compounds compared to CYP17 andsteroid 5α-reductases, the binding to and transactivation of androgenreceptors, and their antiproliferative effects against two humanprostate cancer cell lines, LNCaP and LAPC-4 were studied. Thepharmacokinetics of compounds 5 and 6 were evaluated in mice and the invivo antitumor activities against human LAPC-4 prostate carcinoma werealso evaluated in mice. To our knowledge, all the compounds describedhere, with the exception of compound 15 represent novel entities (Haidaret al., “Novel steroidal pyrimidyl inhibitors of P450 17(17α-hydroxylase/C17-20-lyase)”, Arch. Pharm. Med. Chem., 2001, 334,373-374; and Haidar et al., “Effects of novel17α-hydroxylase/C17,20-lyase (P45017, CYP17) inhibitors on androgenbiosynthesis in vitro and in vivo”, J. Steroid Biochem. Molec. Biol.,2003, 84, 555-562).

The preparation of the new 17-benzoazoles and 17-diazines is outlined inSchemes 1 and 2, respectively. These methods can be applied analogouslyto other analogs described herein.

The key intermediate in our synthesis of the 17-benzazoles,30-acetoxy-17-chloro-16-formylandtrosta-5,16-dine (2) was obtained from(1) by our routine procedure as previously described (Njar et al.,“Nucleophilic vinylic “addition-elimination” substitution reaction of3β-acetoaxy-17-chloro-16-formylandrosta-5,16-diene: A novel and generalroute to 17-substituted-Δ¹⁶-steroids. Part 1. Synthesis of novel17-azolyl-Δ¹⁶ steroids; inhibitors of 17α-hydroxylase/17,20-lyase(P450_(17α))”, Bioorg. Med. Chem. Lett., 1996, 6, 2777-2782; and “Novel17-azolyl steroids; potent inhibitors of cytochrome P45017α-hydroxylase/17,20-lyase (P450_(17α)): Potential agents for thetreatment of prostate cancer”, J. Med. Chem., 1998, 41, 902-912).Treatment of 2 with benzimidazole in the presence of K₂CO₃ in DMF atapproximately 80° C. gave the desired 3β-acetoxy-17-1H-benzimidazole 3in near quantitative yield. Compound 3 was smoothly deformylated with10% palladium on activated charcoal in refluxing benzonitrile to givecompound 4 in 93% yield, from which hydrolysis gave the required3β-hydroxy 17-benzimidazole 5. Modified Oppenauer oxidation of 5afforded the corresponding Δ⁴-3-oxo analog, 6.

The reaction of 2 with benzotriazole in the presence of K₂CO₃ in DMF atapproximately 80° C. gave the desired3β-acetoxy-17-benzo-1H-1,2,3-triazole 7b in excellent yield, togetherwith the 2H-1,2,3-triazole regioisomer 7a in approx 5% yield. These tworegioisomers were readily separated by flash column chromatography (FCC)on silica gel and were also easily identified by their respective protonNMR spectra. Thus, the four aromatic protons of the symmetrical2H-1,2,3-triazole 7a appeared as two pairs of doublets at δ 7.43, 7.45,7.88 and 7.90 while the four aromatic protons of the unsymmetrical1H-1,2,3-triazole 7b appeared as multiplet at δ 7.46 (2H) and doubletsat δ 7.57 (1H) and 8.15 (1H), respectively. In addition, the 16-CHOproton in 7a was significantly shifted downfield to δ 10.66 compared tothat in 7b at δ 9.59. Deformylation of 7b with in situ generation ofRh(1,3-bis(diphenylphosphino)propane)₂ ⁺Cl⁻ catalyst [Rh(dppp)₂ ⁺Cl⁻] inrefluxing xylenes gave compound 8, and following hydrolysis of the3β-acetoxy group, we obtained the target3β-hydroxy-17-(benzo-1H-1,2,3-triazol-1-yl)androsta-5,16-diene (9) in90% yield. Oxidation of 9 afforded 10 in good yield.

Synthesis of the 17-diazines, (17-diazine 14 and 17-pyrimidine 15)commenced from the readily available dehydroepiandrosterone (11, Scheme2), which was converted to the corresponding 17 hydrazone 12 bytreatment with hydrazine hydrate and hydrazine sulfate as previouslydescribed by Potter et al., A convenient, large-scale synthesis ofabiraterone acetate [3β-acetoxy-17(3-pyridyl)androsta-5,16-diene], apotential new drug for the treatment of prostate cancer. Org. Prep.Proc. Int., 1997, 29, 123-128. Treatment of 12 with iodine in thepresence of 1,1,3,3-tetramethylguanidine gave the vinyl 17-iodide 13 inexcellent yield. The palladium catalyzed cross-coupling reactions(Choshi et al., “Total synthesis of Grossularines-1 and -2.” J. Org.Chem., 1995, 60, 5899-5904) of 13 with (2-tributylstannyl)pyrazine or(5-tributylstannyl)pyrimidine proceeded to give3β-hydroxy-17-(2-pyrazyl)-androsta-5,16-diene (14, 15%), and3β-hydroxy-17-(5-pyrimidyl)-androsta-5,16-diene (15, 10%), respectively.The low yields of these two cross-coupling reactions may be due toinstability of the stannyldiazine reagents under the reaction conditionsemployed. The structures of the target compounds, 14 and 15 were readilyidentified by their proton NMR spectra: The three nonequivalent protonsof the 17-pyrazine moiety in 14 appeared as three singlets at δ 8.35,8.48 and 8.70, while for the three protons of the 17-pyrimidine moietyin 15, two equivalent protons appear as a singlet at δ 8.73 and oneproton appeared at δ 9.07. Furthermore, the 17-diazine groups of 14 and15 exhibit different influences on the chemical shifts of theirrespective 16-olefinic protons with respect to the 16-proton of theprecursor Δ⁶-17-iodide 13: the 16-H in 14 appeared as a singlet at δ6.77, being significantly deshielded compared to the 16-H in 13 (δ6.14); the 16-H in 15 appeared at δ 6.11, similar to 13. As indicatedabove, compound 15 was previously reported Haidar et al., however, itwas synthesized by a procedure that is different from the one describedherein.

A representative sample of the novel compounds were then subjected toextensive in vitro and in vivo studies as described in detail in thefollowing sections.

The present invention also relates to method of treating prostate canceror prostate hyperplasia comprising administering to a subject in needthereof an effective amount of a compound in accordance with the presentinvention. The term “treating” is used conventionally, e.g., themanagement or care of a subject for the purpose of combating,alleviating, reducing, relieving, improving, etc., one or more of thesymptoms associated with the prostate disease. Examples of prostatediseases that can be treated include, e.g., prostatic hyperplasia (BPH),and prostate cancer (e.g., prostatic adenocarcinoma).

The specific dose level and frequency of dosage may vary, depending upona variety of factors, including the activity of the specific activecompound, its metabolic stability and length of action, rate ofexcretion, mode and time of administration, the age, body weight, healthcondition, gender, diet, etc., of the subject, and the severity of theprostate cancer or hyperplasia. Any effective amount of the compound canbe administered, e.g., from about 1 mg to about 500 mg per day, morespecifically about 50 mg to about 150 mg per day. The compounds can beadministered in any form by any effective route, including, e.g., oral,parenteral, enteral, intraperitoneal, topical, transdermal (e.g., usingany standard patch), ophthalmic, nasally, local, non-oral, such asaerosol, spray, inhalation, subcutaneous, intravenous, intramuscular,buccal, sublingual, rectal, vaginal, intra-arterial, and intrathecal,etc. The composition may be in a unit dosage form. Typical unit dosageforms include tablets, pills, powders, solutions, suspensions,emulsions, granules, capsules, suppositories, injectable solutions andsuspensions. A compound of the present invention can be administeredalone, or in combination with any other ingredient(s), active orinactive, for example, with physiologically acceptable vehicles to makesuitable pharmaceutical compositions.

The entire disclosure of all applications, patents and publications,cited herein and of U.S. Provisional Application No. 60/657,390, filedMar. 2, 2005, is incorporated by reference herein.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and, all parts and percentages areby weight, unless otherwise indicated.

EXAMPLES Biological Studies

CYP17 Inhibition Studies: A CYP17 inhibition assay is performedaccording to our previously reported procedure, in which intactcytochrome P450c17-expressing E. coli is used as the enzyme source(Grigoryev et al., “Cytochrome P450c17-expressing Escherichia coli as afirst-step screening system for 17α-hydroxylase-C17,20-lyaseinhibitors”, Anal. Biochem.; 1999, 267,319-330; and “Effects of new17α-hydroxylase/C17,20-lyase inhibitors on LNCaP prostate cancer cellgrowth in vitro and in vivo”, Br. J. Cancer, 1999, 81, 622-630. IC₅₀values of the compounds are determined from dose-response curves and arelisted in Table 1. The IC₅₀ values for ketoconazole, abiraterone (aCYP17 inhibitor in clinical trials (O'Donnell, above), Chart 1) and3β-hydroxy-17-(1H-imidazole-1-yl)androsta-5,16-diene (VN/85-1, compound16, Chart 1, believed to be the most potent CYP17 inhibitor (Njar etal., Current Pharm. Design, 1999, 5: 163-180; and J. Med. Chem., 1998,41, 902-912, above) are also determined in the same assay system forcomparison. Some of the new 17-heterocycles exhibit potent inhibition ofCYP17 with IC₅₀ values of 300-915 nM. The benzimidazoles, 5 and 6 are 4-to 6-fold more potent than the benzotriazoles 9 and 10. This resultsuggests that the electronic nature of the 17-heterocycle influenceinhibitory activity. Furthermore, compounds with the Δ⁵-3β-olfunctionality, 5 and 9 are at least 3-fold more potent than thecorresponding analogs with Δ⁴-3-one functionality, 6 and 10,respectively. These results are in contrast to our previous results forthe simple 17-azole CYP17 inhibitors. In that series of inhibitors,there is no marked difference in the inhibitory potencies between theΔ⁵-3β-ol azoles and the corresponding Δ⁴-3-one analogs (Njar et al., J.Med. Chem., 1998, 41, 902-912, above). A possible explanation is thatthe bulkier benzoazoles bind differently at the active site of theenzyme such that the interaction(s) of the moiety at the 3-position isimportant for binding.

The binding of the substrate or inhibitory ligands to the heme componentof some P450 cytochromes is investigated using UV-vis differencespectroscopy (Jefcoat C. R., “Measurement of substrate and inhibitorbinding to microsomal cytochrome P450 by optical differencespectroscopy”, Methods Enzymol., 1978, 52, 258-279). This approach isextended following standard procedure previously reported by us (Njar etal., Bioorg. Med. Chem. Lett., 1996, 6, 2777-2782; and J. Med. Chem.,1998, 41, 902-912). Compounds 5 and 9 each induce a type II differencespectrum, indicating coordination of steroidal nitrogen (N-3 ofbenzimidazole or benzotriazole ring) to the heme iron of CYP17, withformation of low-spin iron. The peak positions for the Soret maximum forthe enzyme complex with 5 and 9 (426 nM) is in agreement with availabledata for the binding of nitrogen ligands to CYP systems, and is also inagreement with our results with other 17-azolyl CYP17 inhibitors (Njaret al., Bioorg. Med. Chem. Lett., 1996, 6, 2777-2782; and J. Med. Chem.,1998, 41, 902-912). The interaction of the benzoazole nitrogen with theheme iron of CYP17 suggests bulk tolerance at the 17-position, becausethe binding affinities of 5 and 9 are identical to that of the lesssterically demanding 16, with a 17-imidazole group.

Of the two 17-diazines tested, the 17-pyrimidine 15 with an IC₅₀ valueof 500 nM is about 8-fold more potent than the 17-pyrazine 14 (IC₅₀=3810nM). As with the benzoazoles, this result suggests that the electronicnature of the 17-heterocycle influence inhibitory activity. Finally,IC₅₀ values in the same assay system for ketoconazole, and abirateroneare evaluated (Table 1). The most potent compound in this series,17-benzimidazole 5, exhibits about 4 and about 3-fold improvements inCYP17 inhibition over these compounds, respectively, although it is lesspotent than 16.

Inhibition of human 5α-reductase isozymes type 1 and 2 in vitro: On thebasis of previous findings that some CYP17 inhibitors are able toinhibit human 5α-reductase enzymes, we briefly evaluated this new seriesof CYP17 inhibitors. The inhibitory activities of compounds 5, 6, 9, 10and finasteride as a reference are determined using the DU-145 cell line(human type 1 enzyme) and human homogenates of BPH tissue (human type 2enzyme) as described by Hartmann et al., “Synthesis and evaluation of2′-substituted 4-(4′-carboxy- or4′-carboxymethylbenzylidene)-N-acylpiperidines: Highly potent and invivo active steroid 5α-reductase type 2 inhibitors”, J. Med. Chem.,2002, 45, 3406-3417. The IC₅₀ values or the percent inhibition values ata concentration of 10 μM for some compound are presented in Table 1.Only compound 6 exhibits potent inhibition of both type 1 and 2 enzymes(IC₅₀=770 and 480 nM, respectively), although it is several fold lesspotent than finasteride (IC₅₀=60 and 2 nM, respectively).

LNCaP and PC-3AR androgen receptor binding assays: Because we hadpreviously demonstrated that some of our CYP17 inhibitors are potentantiandrogens for both the mutant and wild-type AR (Long et al.,Gregoriyev et al. and Njar et al., J. Med. Chem., 1998, 41, 902-912,above) it was of interest to assess the ability of this series of CYP17inhibitors to bind to these receptors. AR competition is determinedusing labeled R1881 ([³H]-R1881) in the androgen-sensitive LNCaP cells,that express mutant AR, and the androgen-independent PC-3 cells stablytransfected with the wild-type AR (designated PC-3AR). Compounds 5, 6,14 and 15, in the nanomolar concentration range, compete effectivelywith labeled R1881 for binding to both types of ARs in a dose-dependentmanner (Figure not shown). Compounds 5, 6, 14 and 15, with IC₅₀ valuesof 384, 242, 336 and 374 nM, respectively (Table 1), versus the wildtype AR are 29 to 45-fold more potent than with the clinically usedantiandrogen, flutamide (IC₅₀=10,985 nM). As shown in Table 1, thebinding affinities for the mutant AR of 5 and 6 are comparable to thatof casodex, a currently used antiandrogen, but again superior to that offlutamide. However, the biological activity of flutamide is derivedmainly from a metabolite, hydroxyflutamide, which is a much more potentAR antagonist.

Effects of agents on LNCaP mutant AR-mediated transcription: Next, weasked whether compounds 5 and 6 are acting as AR agonists orantagonists. A study on androgen-regulated transcriptional activation isperformed in LNCaP cells transiently transfected with a probasinluciferase reporter construct AARZ-Luc (luciferase activity assay) (Kimet al., “Synergism of cytoplasmic kinases in IL6-inducedligand-independent activation of androgen receptor in prostate cancercells”, Oncogene, 2004, 23:1838-1844; and Zhang et al., “A Smallcomposite probasin promoter confers high levels of prostate-specificgene expression through regulation by androgens and glucocorticoids inVitro and in Vivo”, Endocrinology, 2000, 141: 4698-4710). Compounds 5, 6or casodex each at 0.1 and 10.0 μM have no effect on luciferaseactivity, whereas luciferase expression is increased approximately99.6-fold after treatment with 1.0 nM DHT for 18 h (FIG. 1).Furthermore, luciferase expression induced by exposure to 1.0 nM DHT isdecreased in a concentration-dependent manner by 5, 6, and casodex andin a similar fashion (FIG. 1). Together, these results suggest thatcompounds 5 and 6 like casodex do not possess AR agonistic or partialagonistic activity and may be considered as strong, pure androgenantagonists. Although we did not test the compounds with PC-3AR/LUcells, which express wild-type AR, it is likely that they may alsobehave in a similar fashion. We has previously shown that some of ourCYP17 inhibitors were more comparable to casodex than to flutamide (Longet al., above), and this appears to be the case with these newcompounds. In general, our novel compounds interact strongly with bothAR types, an indication that the compounds may be useful for thetreatment of patients with tumors expressing either wild-type or mutatedAR, or for patients with amplified AR expression.

Effects of benzoazoles on the growth of LNCaP and LAPC-4 prostate cancercells in vitro: The abilities of compounds 5 and 6 to inhibitproliferation in mutant LNCaP cells stimulated by 1 nM DHT is examined.This concentration of DHT stimulated LNCaP cell proliferation by about2-fold compared to vehicle-treated cells (FIG. 2A). As shown in FIG. 2A,compounds 5 and 6 each inhibit the DHT-induced LNCaP cell proliferationin a dose-dependent fashion, with IC₅₀ values of 6.0 and 1.8 μM,respectively. Casodex is used as a positive control, and it exhibitssimilar inhibition of DHT-induced LNCaP cell proliferation (FIG. 2A,IC₅₀=8.6 μM). Treating the androgen-sensitive LAPC4 prostate cell linewith 10 nM DHT, surprisingly, does not significantly induce cellproliferation (FIG. 2B). Other investigators have also reported that theresponse of LAPC4 cells to androgens is not as pronounced as thatobserved in LNCaP cells (Thompson et al., “Androgen antagonist activityby the antioxidant moiety of vitamin E,2,2,5,7,8-pentamethyl-6-chromanol in human prostate carcinoma cells”,Molec. Caner Thera., 2003, 2, 797-803). However, compounds 5, 6 andcasodex each exhibit a dose-dependent inhibition of this cell line (FIG.2B) as with the LNCaP cells. The order of inhibitory potency of LAPC4cell proliferation is 6>5>casodex, with IC₅₀ values of 1.0, 3.2 and 10μM, respectively. Together, these results suggest that 5 and 6 may beacting to block the action of DHT in stimulating cell proliferation, incorrelation with their androgen receptor binding and activationproperties described above. Compounds 5 and 6 are amongst the mostpotent antiandrogens described to date.

Pharmacokinetics of 5 and 6 and metabolism of 5: The pharmacokineticproperties in male SCID mouse for the two lead compounds, 5 and 6 arestudied following our recently described procedure for other CYP17inhibitors (Nnane et al., “Pharmacokinetic profile of3β-hydroxy-17-(1H-123-triazol-1-yl)androsta-5,16-diene (VN/87-1), apotent androgen synthesis inhibitor in mice”, J. Steroid Biochem. Molec.Biol., 2001, 71, 145-152; and Handratta et al., “Potent CYP17inhibitors: improved syntheses, pharmacokinetics and anti-tumor activityin the LNCaP human prostate cancer model”, J. Steroid Biochem. Molec.Biol., 2004, 92, 155-165. The results are summarized in Table 2 andFIGS. 3-5.

On reverse phase HPLC, 5 [retention time (rt)=21.6 min] is well resolvedfrom the internal standard (16, rt=11.5 min), a metabolite (rt=17.3 min)and other endogenous compounds in mouse plasma (FIG. 3). The calibrationcurves derived for 5 are linear and reproducible (data not shown), theinter- and intra-assay variability is less than 10% and its limit ofdetection is 100 ng/ml. The HPLC assay is validated and used to monitor5 in mice plasma.

Following subcutaneous administration, the plasma concentration of 5declines exponentially with a mean half-life of about 44.17 min andelimination rate constant of 56.5 min⁻¹. Compound 5 is cleared at a rateof 1986.14 ml/h/kg from the systemic circulation and was not detected 6h after administration. The calculated non-compartmental pharmacokineticparameters based on the plasma concentration profile followingsubcutaneous administration of 5 are shown in Table 2. The plasmaconcentration-time curves after s.c. administration of 5 (50 and 100mg/kg) to male SCID mice are also shown in FIG. 4. After s.c.administration of 5, the observed plasma concentration in mice reachpeak levels 30.0 min post dose. Compound 5 is well absorbed from thesubcutaneous site and the area under the curve for the plasmaconcentration versus time profiles after s.c. administration increasesproportionately to dose as the administration dose is changed from 50 to100 mg/kg. Furthermore, the elimination half-life, and mean residencetime are relatively constant as the dose of 5 increases from 50 to 100mg/kg (Table 1). These results indicate that the pharmacokinetic profileof 5 is dose independent.

FIG. 5 shows that a significant amount of a polar metabolite [retentiontime, 17.3 min, see FIG. 3)] is formed from 5 and present in the plasmaduring the in vivo pharmacokinetic studies. The maximum amount of themetabolite is 67.72%, attained about 2 h post dose. This metaboliteshows identical retention time as compound 6. This metabolite istentatively identified by LC-MS; its molecular mass (m/z 391=M+H⁺) isconsistent with the structure of 3-oxo-Δ^(5,16)-tetrahydro compound 5(i.e., 17-(1H-benzimidazol-1-yl)androst-3-one). The metabolite may havebeen formed from 5 via oxidation of the 3β-OH→3-oxo, followed byreduction (reductases) of both Δ⁵ and Δ¹⁶ double bonds. A similarmetabolite was previously identified (formed as a result of oxidation ofa 3β-OH→3-oxo, followed by isomeraization of Δ⁵ double bond) in malemice of a closely related steroidal 17-imidazole (Handratta et al.,above).

A major metabolite of 5, i.e., 17-(1H-benzimidiazol-1-yl)androst-3-one,may be synthesized from trans-androsterone; see Scheme 3. It is alsoexpected to have analogous activity.

The in vivo pharmacokinetics of 6 in mice is unlike that of compound 5due to the relatively low C_(max) and significantly higher eliminationrate (FIG. 4 and Table 2). In addition, we did not detect anymetabolism(s) of compound 6 in the plasma, in contrast to ourobservation with compound 5.

Effects of 5 and 6 on LAPC4 Xenografts grown in SCID mice: On the basisof impressive multiple in vitro biological activities, i.e., potentinhibition of CYP17, strong antiproliferative prostate cancer cellactivity and antiandrogenic activities, 5 and 6 are selected for in vivoantitumor efficacy studies in androgen-depended LAPC4 human prostatecancer xenograft model.

In the first experiment, the effect of compounds 5 and 6 on the growthof well-established LAPC4 prostate cancer tumors in SCID mice isdetermined, and castration is used as the reference treatment.Tumor-bearing mice are assigned (n=5/group) to receive one of two dosesof 5 or 6 (0.15 mmol/kg once-daily or 0.15 mmol/kg twice-daily). Tumorvolumes are measured weekly and compared with controls receiving vehicleor castrated mice.

Castration leads to a 55% reduction of final tumor volume, as comparedto the control (FIG. 6). Administration of 0.15 mmol/kg once daily and0.15 mmol/kg twice daily of 5 results in reduction of average finaltumor volumes of 41% and 86.5%, respectively, compared to tumors invehicle-treated control animals (FIG. 6). In contrast to the excellenttumor growth inhibition for 5 treated mice, mice treated with compound 6are either ineffective at the low dose or even show stimulation of tumorgrowth compared to control (FIG. 6). The inability of 6 to inhibit LAPC4tumor growth in vivo is especially disappointing because the compound isvery effective at inhibiting PCA cell growth in vitro, and is a highlypotent pure antiandrogen (see FIG. 1). The highly significant disparityin the in vivo antitumor efficacy of 5 and 6 cannot easily beattributable to differences in the pharmacokinetic properties of the twocompounds. The underlying reason(s) for the dramatic differences in invivo antitumor efficacy of these two closely related compounds isunknown at this time. However, it may be attributable to 6 beingconverted in the animals to metabolite(s) that may be a strong agonistof androgen receptor thus causing tumor growth stimulation. During thestudy, all mice were weighed once per week. The body weights of alltreated groups increased slightly and were similar to the increaseobserved with the control group. All mice appeared healthy and noadverse effects were observed suggesting that the compounds were withoutsignificant toxicity.

The second in vivo experiment tests the ability of 5 to inhibit thegrowth of LAPC4 prostate cancer cells growing in SCID mice, and 16(Chart 1), a previously identified potent CYP17 inhibitor/antiandrogen(Gregoriyev et al. and Njar et al., J. Med. Chem., 1998, 41, 902-912,above) and castration are used as reference treatments. In thisexperiment, treatment begins on the day that mice were inoculatedsubcutaneously with hormone-dependent LAPC4 cells and are castrated orinjected sc twice daily with 5 or 16. FIG. 7 shows the effects of thevarious treatments on the emergence and on the size of tumors during the21 weeks of therapy.

All other groups develop palpable and measurable tumor at week 10 oftherapy except for the group treated with 16 (0.15 mmol/kg twice-daily)that develop palpable and measurable tumors at week 11. Total tumorvolume in the control mice increases by 8-fold over 14 weeks oftreatment when mice are sacrificed because of the large tumors. Thus,the tumor volumes for the other groups are compared to those of thecontrol group at week 14 of treatment. Tumor volume in the castratedmice increases by only 4.1 fold (about 50% reduction compared tocontrol), and is similar to the 3.7 fold increase (53.8% reductioncompared to control) observed in mice treated with 16. In the micetreated with 5 (0.15 mmol/kg twice-daily), tumor volume increases byonly 0.5 fold, which represents a 93.8% reduction versus control mice(P=0.00065). At week 16, the mean tumor volume in the compound 5-treatedanimals is found to be lower (almost negligible and dormant) than theirmean tumor volume at week 10 when measurable tumors emerge. Furthermore,5 causes a significant inhibitory effect on tumors, compared to 16 orcastration, P=0.005 and 0.05, respectively. In general, tumors in thecontrol, castration and compound 16 treated mice grow rapidly, while thetumor of the 5 treated mice grows very slowly and in a biphasic manner(FIG. 7). Compound 5 is the most effective agent, and significantly ismuch more effective than castration at inhibition of tumor growth. It isinteresting to note that although 16 is 6 times more potent than 5 inCYP17 inhibition, the latter exhibits a superior in vivo antitumoractivity. The reason(s) responsible for this phenomenon is unknown atthis time, but may be in part due to better pharmacokinetic and orpharmacodynamic properties of 5.

To determine whether the “dormant” compound 5-treated prostate tumors(see FIG. 7, week 16) are able to grow on a lower dose of 5, its dosewas reduced to 0.15 mmol/kg thrice a week (a 78.6% reduction in dosage)from weeks 16-19, and the tumor volumes measured weekly. During thisperiod of treatment with reduced dose of the compound, tumors resumegrowth (FIG. 7). After this 3-week interval, drug treatment with theusual dose is resumed, and the tumor growth slows and reaches a plateau.These data suggest a cytostatic nature of this treatment and infer theneed for continuous administration to achieve the antitumor effect.

At the end of the experiment, the levels of 5 in the tumors and organsof the 5-treated mice are determined. The 5 levels by HPLC in thetumors, testis and liver 1 h after administration of the final dose(insert of FIG. 7) are measured. Interestingly, a small (˜15% relativeto 5) amount of metabolite is detected only in the liver tissues. Thismetabolite has the same retention time as the metabolite observed in theplasma (vide supra). The highest concentration of 39.0±8.4 μg/mg tissueof 5 is measured in the s.c. tumors. The concentrations in the liver andtestis are lower but detectable. The level of 5 in tumors issignificantly higher that the levels measured in the plasma, which maybe a result of accumulation of the compound through the period of theexperiment. Thus, inhibition of tumor growth by 5 can be explained inpart higher concentrations in tumor xenografts, which may exert directcytotoxic/cytotastic effect on the prostate cancer cells. It should bestated that there is evidence to suggest a possible direct cytotoxiceffect of ketoconazole (a modest CYP17 inhibitor) on prostate cancercells.³³ In addition; the accumulation of 5 in the testes would enableinhibition of testosterone synthesis in the animals.

Although it is well established that LAPC4 are androgen-dependent, thesecells can become androgen-independent, and as such represent a suitablemodel that mimics prostate cancer development in patients (Chen et al.,above, and Kline et al., “Progression of metastatic human prostatecancer to androgen independence in immunodeficient SCID mice.” Nat.Med., 1997, 3, 402-408). As shown in FIG. 7, we are able to replicatethis phenomenon. Furthermore, our results show that treatment with 16 orcastration effectively suppresses tumor growth for a certain period(androgen-dependent phase), but was ineffective thereafter (possibly asa result of an androgen-independent phase) since the tumors grow rapidlyjust as in intact control mice. Tumor growth in the mice treated with 5is strongly suppressed throughout the treatment period. This suggeststhat 5 may have effects on androgen-independent prostate cancer.However, it is also plausible that treatment with this compound enablesLAPC4 tumors to remain androgen dependent for a longer period andtherefore responsive to antiandrogen therapy.

Recent studies that clearly demonstrate the up-regulation andinvolvement of AR in advanced and recurrent PCA (Mohler et al., and Chenet al., above) have renewed interest in the androgen receptor as atarget for development of drugs to treat PCA (Tindall et al., “Symposiumon androgen action in prostate cancer”, Cancer Res., 2004, 64,7178-7180). Because of its potent properties, 5 may be an excellentcandidate.

Conclusions:

The data reinforce our earlier concept of modification of the C17substituent of Δ¹⁶ steroids to produce potent inhibitors of CYP17 aswell as potent AR antagonists. The 17-benzimidazoles 5 and 6 are shownto coordinate the heme iron of CYP17, a property that may in part beresponsible for their enzyme inhibitory activity. Compounds 5 and 6exhibit almost equipotent in vitro activities for CYP17 inhibition, ARantagonism, and inhibition of prostate cancer cell growth. Surprisingly,the compounds are very different in their antitumor activities, as 5causes marked suppression of LAPC4 tumor xenograft growth, and incontrast, 6 (0.15 mmol/kg twice daily) enhances tumor growth. Thepresent study provides compelling evidence that 5 is a potent inhibitorof human prostate tumor growth and is remarkably more effective thancastration. This is the first example of a CYP17 inhibitor/antiandrogendemonstrating in vivo antitumor activity against a prostate cancer tumorto an extent that is superbly more effective than castration. Theseimpressive biological activities, makes 5 a strong candidate for furtherdevelopment as a potential drug for the treatment of prostate cancer inhumans. The excellent antitumor activity of compound 5, containing abenzimidazole group makes the benzimidazoles a preferred group. However,analogs of 5 as discussed above are expected to have related activityand are included in the invention.

Experimental Section

Chemistry: General procedures and techniques were identical with thosepreviously reported (Njar et al., J. Med. Chem., 1998, 41, 902-912).Infra red spectra are recorded on a Perkin Elmer 1600 FTIR spectrometerusing solutions in CHCl₃. High-resolution mass spectra (HRMS) aredetermined on a 3-Tesla Finnigan FTMS-2000 FT mass spectrometer, ESImode (Ohio State University, Department of Chemistry). As a criterion ofpurity for key target compounds, we provided high resolution massspectral data with HPLC chromatographic data indicating compoundhomogeneity. Low-resolution mass spectra (LRMS) are determined on aFinnegan LCR-MS. Melting points (mp) are determined with a Fischer Johnsmelting point apparatus and are uncorrected. Dehydroepiandrosterone anddehydroepiandrosterone acetate were purchased from Aldrich, Milwaukee,Wis. 5-Tributylstannylpyrimidine and 2-tributylstannylpyrazine werepurchased from Frontier Scientific, Inc., Logan, Utah.

3β-Acetoxy-17-chloro-16-formylandrosta-5,16-diene (2): This compoundprepared from 30-acetoxyandrost-5-en-17-one (1) as previously described,provided spectral and analytical data as described (Njar et al., J. Med.Chem., 1998, 41, 902-912).

3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-formylandrosta-5,16-diene (3): Amixture of 3β-Acetoxy-17-chloro-16-formylandrosta-5,16-diene (2, 2.5 g,6.65 mmol), benzimidazole (2.35 g, 19.9 mmol), and K₂CO₃ (2.76 g, 23.9mmol) in dry DMF (20 mL) is stirred at ca. 80° C. under Ar for 1.5 h.After cooling to room temperature, the reaction mixture is poured ontoice-cold water (250 mL) and the resulting precipitate is filtered,washed with water, and dried to give a crude dirty white solid (ca. 2.9g). Purification by FCC [petroleum ether/EtOAc/Et₃N (6:4:0.3)] gives 2.7g (88.7%) of pure compound 3: mp 227-230° C.; IR (CHCl₃) 3691, 3024,2951, 2359, 1725, 1670, 1604, 1491, 1452, 1375, 1253, 1032, 897, 852,818, 700, 657, 618, 576, 565, 550, 529, 511, 476 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ 1.07 (s, 6H, 18- and 19-CH₃), 2.04 (s, 3H, 3β-OCH₃), 4.60 (m,1H, 3α-H), 5.43 (br s, 1H, 6-H), 7.35 (br. s, 2H, aromatic-Hs), 7.85 (s,1H, aromatic-H), 7.98 (s, 1H, aromatic-H), 7.98 (s, 1H, 2¹-H) and 9.59(s, 1H, 16-CHO). HRMS calcd 481.2462 (C₂₉H₃₄O₃N₂.Na⁺), found 481.2454.

3β-Acetoxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (4): A solutionof 3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-formylandrosta-5,16-diene (3,2.04 g, 4.45 mmol) in dry benzonitrile (10 mL) was refluxed in thepresence of 10% palladium on activated charcoal (1.02 g, i.e., 50%weight of 3) for 5 h. After cooling to room temperature, the catalystwas removed by filtration through a Celite pad. The filtrate wasevaporated, and the residue was purified by FCC [petroleumether/EtOAc/Et₃N (7.5:3:0.5)] gave 1.41 g (73.8%) of pure compound 4: mp159-160° C.; IR (CHCl₃) 3687, 2947, 2854, 2358, 2340, 1725, 1633, 1609,1557, 1489, 1454, 1373, 1291, 1253, 1195, 1136, 1031, 985, 910, 839,735, 665, 590, 544, 533, 513, 502, 488 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ1.02 (s, 3H, 18-CH₃), 1.07 (s, 3H, 19-CH₃), 2.04 (s, 3H, 3β-OCH₃), 4.62(m, 1H, 3α-H), 5.43 (br s, 1H, 6-H), 5.98 (s, 1H, 16-H), 7.30 (m, 2H,aromatic-Hs), 7.49 (s, 1H, aromatic-H), 7.81 (s, 1H, aromatic-H), and7.95 (s, 1H, 2¹-H). HRMS calcd 453.2512 (C₂₈H₃₄O₂N₂.Na⁺), found453.2511.

3β-Hydroxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (5): The acetate4 (1.3 g 3.02 mmol) was dissolved in methanol (20 mL) under an inert Aratmosphere, and the resulting solution treated with 10% methanolic KOH(8 mL). The mixture was stirred at room temperature for 1.5 h, and thenconcentrated under reduced pressure at approx. 40° C. to a volume of 10mL. This solution was poured into ice water (300 mL), and the resultingwhite precipitate was filtered, washed with water and dried.Crystallization from EtOAc/MeOH gave 5 (1.10 g, 94%), mp 189-190° C.; IR(CHCl₃) 2934, 2339, 1609, 1490, 1453, 1291, 1040, 837, 808, 705, 663,608, 578, 550, 517 cm⁻¹; ¹HNMR (300 MHz, CDCl₃) δ 1.02 (s, 3H, 18-CH₃),1.07 (s, 3H, 19-CH₃), 3.55 (m, 1H, 3α-H), 5.41 (br s, 1H, 6-H), 5.99 (s,1H, 16-H), 7.30 (m, 2H, aromatic-Hs), 7.54 (s, 1H, aromatic-H), 7.80 (s,1H, aromatic-H), and 7.96 (s, 1H, 2¹-H). HRMS calcd 411.2407(C₂₆H₃₂ON₂.Na⁺), found 411.2396.

17-(1H-benzimidazol-1-yl)androsta-4,16-diene-3-one (6): From a mixtureof compound 5 (660 mg, 1.70 mmol), 1-methyl-4-piperidone (2.5 mL), andtoluene (40 mL) was distilled off ca. 10 mL. Aluminum isopropoxide (521mg, 2.55 mmol) was then added, and the mixture was refluxed under Ar for4 h. After cooling, the mixture was diluted with EtOAc (50 mL), washedsuccessively with 5% aqueous NaHCO₃ (×3) and brine (×2), and then dried(Na₂SO₄). The solvent was evaporated, and the crude product was purifiedby FCC [CH₂Cl₂/EtOH (25:1)] to give the title compound 6 (544 mg, 82%):mp 201-204° C.; IR (CHCl₃) 2946, 2858, 1622, 1611, 1490, 1453, 1376,1291, 1270, 1228, 1189, 893, 850, 837, 722, 662, 615, 568, 553, 537, 519cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 1.04 (s, 3H, 18-CH₃), 1.24 (s, 3H,19-CH₃), 5.78 (s, 1H, 4-H), 5.99 (s, 1H, 16-H), 7.31 (m, 2H,aromatic-Hs), 7.48 (m, 1H, aromatic-H), 7.81 (s, 1H, aromatic-H), and7.95 (s, 1H, 2¹-H). HRMS calcd 409.2250 (C₂₆H₃₀₀N₂.Na⁺), found 409.2250.

Reaction of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene (2) withbenzo-1H-1,2,3-triazole and K₂CO₃:30-Acetoxy-17-(benzo-2H-1,2,3-triazol-2-yl)-16-formylandrosta-5,16-diene(7a) and3β-Acetoxy-17-(benzo-1H-1,2,3-triazol-1-yl)-16-formylandrosta-5,16-diene(7b): A mixture of compound 2 (2.5 g, 6.65 mmol), benzotriazole (2.35 g,19.9 mmol), and K₂CO₃ (2.76 g, 23.9 mmol) in dry DMF (20 mL) was stirredat ca. 80° C. under Ar for 45 min. After cooling to room temperature,the reaction mixture was poured onto ice-cold water (250 mL) and theresulting precipitate was filtered, washed with water, and dried to givea crude dirty white solid. Purification by FCC [pet. Ether/EtOAc, (4:1)]first gave3β-acetoxy-17-(benzo-2H-1,2,3-triazol-2-yl)-16-formylandrosta-5,16-diene(7a, 0.3 g, 9.8%) as minor product; mp 248-250° C.; IR(CHCl₃) 3023,2945, 2358, 1725, 1657, 1600, 1375, 1257, 1032, 728, 656, 584, 564, 540,526, 506, 498 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 1.11 (s, 3H, 18-CH₃), 1.37(s, 3H, 19-CH₃), 2.04 (s, 3H, 3β-OCH₃), 4.62 (m, 1H, 3α-H), 5.43 (br s,1H, 6-H), 7.43 (d, 1H, J=2.4 Hz, aromatic-Hs), 7.45 (d, 1H, J=2.7 Hz,aromatic-H), 7.88 (d, 1H, J=2.7 Hz, aromatic-H), 7.90 (d, 1H, J=2.4 Hz,aromatic-H) and 10.66 (s, 1H, 16-CHO). HRMS calcd 482.2414(C₂₈H₃₃O₃N₃.Na⁺), found 482.2413. Further elution with the same solventsystem afforded the major product,3β-acetoxy-17-(benzo-1H-1,2,3-triazol-1-yl)-16-formylandrosta-5,16-diene(7b, 2.3 g, 75.4%); mp: 186-188° C.; IR (CHCl₃) 3023, 2948, 1725, 1670,1604, 1488, 1450, 1374, 1253, 1196, 1032, 846, 824, 720, 658, 619, 548,527, 504, 497 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 1.07 (s, 6H, 18- and19-CH₃), 2.04 (s, 3H, 3β-OCH₃), 4.60 (m, 1H, 3α-H), 5.43 (br s, 1H,6-H), 7.46 (m, 2H, aromatic-Hs), 7.57 (d, 1H, J=6.9 Hz, aromatic-H),8.15 (d, 1H, J=8.4 Hz), aromatic-H), and 9.59 (s, 1H, 16-CHO). HRMScalcd 482.2414 (C₂₈H₃₃O₃N₃.Na⁺), found 482.2416.

3β-Acetoxy-17-(benzo-1H-1,2,3-triazol-1-yl)androsta-5,16-diene (8): Amixture of bis(triphenyphosphine)rhodium(I) carbonyl chloride (303 mg,0.438 mmol) and 1,3-bis-(diphenylphosphino)propane (394 mg, 0.954 mmol)in dry xylene (40 mL) was stirred at 80° C. under Ar for 15 min whenfine yellow precipitate formed. Compound 7b (1.71 g, 3.72 mmol) wasadded, and the mixture was refluxed under Ar for 18 h, and thenconcentrated under reduced pressure. The crude product was purified byFCC [pet ether/EtOAc/Et₃N, (8.9:1:0.1)] to give 1.2 g (74.7%) of purecompound 8; mp 184-186° C. IR (CHCl₃) 3063, 2918, 2389, 2358, 1725,1458, 1373, 1254, 1069, 1031, 843, 809, 786, 692, 646, 560, 535, 528,512, 494 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 1.10 (s, 3H, 18-CH₃), 1.25 (s,3H, 19-CH₃), 2.04 (s, 3H, 30-OCH₃), 4.64 (m, 1H, 3α-H), 5.43 (br s, 1H,6-H), 6.01 (s, 1H, 16-H), 7.40 (t, 1H, J=7.8 Hz, aromatic-H), 7.51 (t,1H, J=7.8 Hz, aromatic-H), 7.67 (d, 1H, J=8.1 Hz, aromatic-H), and 8.10(d, 1H, J=8.1 Hz, aromatic-H). HRMS calcd 454.2465 (C₂₇H₃₃O₂N₃.Na⁺),found 454.2469.

3β-Hydroxy-17-(benzo-1H-1,2,3-triazol-1-yl)androsta-5,16-diene (9): Themethod followed that described for compound 5 but using3β-acetoxy-17-(benzo-1H-1,2,3-triazol-1-yl)androsta-5,16-diene (8; 700mg, 1.62 mmol). Recrystallization from EtOAc/MeOH give the titlecompound 9 (600 mg, 95%); mp 241-244° C.; IR (CHCl₃) 3603, 2937, 2859,1609, 1488, 1451, 1373, 1287, 1243, 1069, 1040, 1007, 953, 845, 805,715, 665, 618, 570, 553, 517 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 1.09 (s,3H, 18-CH₃), 1.24 (s, 3H, 19-CH₃), 3.55 (m, 1H, 3α-H), 5.41 (br s, 1H,6-H), 6.06 (s, 1H, 16-H), 7.40 (t, 1H, J=7.8 Hz, aromatic-H), 7.52 (t,1H, J=7.8 Hz, aromatic-H), 7.67 (d, 1H, J=8.1 Hz, aromatic-H), and 8.10(d, 1H, J=8.1 Hz, aromatic-H). HRMS calcd 412.2359 (C₂₅H₃₁ON₃.Na⁺),found 412.2365.

17-(benzo-1H-1,2,3-triazol-1-yl)androsta-4,16-diene-3-one (10): Themethod followed that described for compound 6 but usingβ-hydroxy-17-(benzo-1H-1,2,3-triazol-1-yl)androsta-5,16-diene (9; 500mg, 1.28 mmol). Purification of the crude product by FCC [CH₂Cl₂/EtOH,(50:1)] afforded the titled compound 10 (420 mg, 84.4%); mp: 280-283°C.; IR (CHCl₃) 2944, 1658, 1450, 1070, 8444, 825, 721, 624, 589, 564,554, 541, 521 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 1.26 (s, 3H, 18-CH₃), 1.27(s, 3H, 19-CH₃), 5.77 (s, 1H, 4-H), 6.01 (s, 1H, 16-H), 7.40 (t, 1H,J=7.8 Hz, aromatic-H), 7.52 (t, 1H, J=7.8 Hz, aromatic-H), 7.67 (d, 1H,J=7.8 Hz, aromatic-H), and 8.10 (d, 1H, J=8.1 Hz, aromatic-H). HRMScalcd 410.2203 (C₂₅H₂₉ON₃.Na⁺), found 410.2185.

Dehydroepiandrosterone-17 hydrozone (12): Dehydroepiandrosterone (11,3.5 g, 12.2 mmol) was dissolved in ethanol (60 mL); and the resultingsolution was treated with hydrazine hydrate (2.37 mL, 0.049 mol)followed by a solution of hydrazine sulfate (7.9 mg, 0.061 mmol) in 0.25mL of water. The mixture was stirred at room temperature for 12 h andthen poured into ice water. The resulting precipitate was filtered,washed with water, and dried to give white crystals of the titledcompound 12; mp: 242-244° C. (lit. 204-206° C.);²² ¹H NMR (300 MHz,CDCl₃): δ 0.76 (s, 3H, 18-CH₃), 1.05 (s, 3H, 19-CH₃), 3.74 (br s, 1H,3-H) and 5.35 (s, 1H, 6-H).

17-Iodoandrosta-5,16-diene-3β-ol (13): A stirred solution of iodine(12.16 g, 0.0203 mol) in dry of THF (144 mL) and dry of Et₂O (72 mL) wascooled in an ice bath to 0° C. and the solution was treated with1,1,3,3, tetramethylguanidine (6.72 mL, 6.24 g, 0.054 mole). A solutionof compound 12 (3.0 g, 9.9 mmol) in THF (81 mL) was added dropwise tothe iodine solution over 2 h maintaining the reaction temperature at 0°C. The reaction mixture was then concentrated under vacuum, cooled in anice-bath, and then dried to under vacuum at room temperature to afford ayellow solid (13, 3.65 g, 92.4%). mp: 169-171° C. (lit. 175-176° C.);²²IR(CHCl₃) 2935, 1371, 1039, 862, 843, 799, 715, 665, 582, and 566 cm⁻¹;¹H NMR (300 MHz, CDCl₃): δ 0.76 (s, 3H, 18-CH₃), 1.05 (s, 3H, 19-CH₃),3.50 (br s, 1H, 3α-H), 5.35 (s, 1H, 6-H) and 6.14 (s, 1H, 16-H).

3β-Hydroxy-17-(2-pyrazyl)-androsta-5,16-diene (14): A mixture of17-iodoandrosta-5,16-diene-3β-ol (13; 0.5 g, 1.257 mmol) in solutionwith dry dimethylformamide (DMF, 10 mL) along withtetrakis(triphenylphosphate) palladium (Pd(PPh₃)₄) (71.6 mg, 0.062 mmol)and (2-tributylstannyl) pyrazine (774.6 mg, 2.099 mmol) was heated at120° C. for 20 h. After cooling, the mixture was diluted with cold water(50 mL), and extracted with EtOAc (30 mL×3). The combined EtOAc extractwas washed with brine and water, dried over Na₂SO₄ and then concentratedto give a brownish solid. This crude product was purified by flashcolumn chromatography [FCC, pet.ether/EtOAc/Et₃N (3:2:0.15)] to give 14(66 mg, 15%); mp: 199-201° C. ¹H NMR (300 MHz, CDCl₃): δ 0.94 (s, 3H,18-CH₃), 1.08 (s, 3H, 19-CH₃), 3.52 (br s, 1H, 3α-H), 5.40 (s, 1H, 6-H),6.77 (s, 1H, 16-H), 8.35 (s, 1H, pyrazine-H), 8.48 (s, 1H, pyrazine-H),8.70 (s, 1H, pyrazine-H). HRMS calcd 350.2358 (C₂₃H_(30l ON) ₂), found350.2354.

3β-Hydroxy-17-(5-pyrimidyl)-androsta-5,16-diene (15): Reaction of 13(0.645 g, 1.623 mmol) as described above for 14, but using(5-tributylstannyl) pyrimidine (1.0 g, 2.710 mmol) dissolved in 10 mL ofdry DMF along with (Pd(PPh₃)₄) (92.88 mg, 0.0804 mmol) and(5-tributylstannyl) pyrimidine (1.0 g, 2.710 mmol) and followingpurification by [FCC, pet. ether/EtOAc/Et₃N (3:2:0.15)] gave3β-hydroxy-17-(5-pyrimidyl)-androsta-5,16-diene 15 (44 mg, 10%); mp:231-233° C. (lit. 240-242° C.);¹⁹ ¹H NMR (300 MHz, CDCl₃): δ 1.05 (s,3H, 18-CH₃), 1.08 (s, 3H, 19-CH₃), 3.83 (br s, 1H, 3α-H), 5.39 (s, 1H,6-H), 7.26 (s, 1H, 16-H), 8.73 (s, 2H, 4¹-H and 6¹-H) and 9.07 (s, 1H,2¹-H). HRMS calcd 350.2358 (C₂₃H₃₀ON₂), found 350.2348.

In Vitro Assay of CYP17: The in vitro CYP17 inhibitory activities of thecompounds are evaluated using our rapid acetic acid releasing assay(AARA), utilizing intact P450c17-expressing E. coli as the enzyme source(Grigoryev, above). It involves the use of[21-³H]-17α-hydroxypregnenolone as the substrate and CYP17 activity ismeasured by the amount of tritiated acetic acid formed during thecleavage of the C-21 side chain of the substrate. This establishes thatthe method is comparable in terms of accuracy and reliability to theHPLC analysis procedure used by researchers in the field (Grigoryev,above). IC₅₀ values are obtained directly from plots relating percentageinhibition versus inhibitor concentration over appropriate ranges. Eachcompound is tested at a minimum of five different concentrations. Theassays are performed in triplicate, and the IC₅₀ values reported are themean of triplicate experiments. The standard deviations were ±5% of themean values.

Human 5α-Reductase Type 1 and 2 Assay: The inhibitory activities ofcompounds and finasteride as reference are determined using the DU145cell line (for human type 1 enzyme) and human prostate homogenate (BPHtissue for type 2 enzyme) according to the procedure described byHartmann and colleagues (Picard et al., “Synthesis and evaluation of2′-substituted 4-(4′-carboxy- or4′-carboxymethylbenzylidene)-N-acylpiperidines: Highly potent and invivo active steroid 5α-reductase type 2 inhibitors”, J. Med. Chem.,2002, 45, 3406-3417). The percent inhibition values at a concentrationof 10 μM or, in case of more potent compounds, the IC₅₀ values aredetermined.

Competitive Androgen Receptor (Ar) Binding and Luciferase Assays:

AR Binding/Competition Assay: Wells in 24-well multiwell dishes arecoated with poly-1-lysine (0.05 mg/ml) for 5 minutes, dried, rinsed withsterilized, distilled, water, and dried for 2 hours. To determine thekinetics of R1881 binding to the LNCaP AR and the wild-type AR, LNCaPand PC3AR cells are plated (2−3×10⁵) in 24 well multiwell dishes insteroid-free medium and allowed to attach. The following day the mediumis replaced with serum-free, steroid free RPMI supplemented with 0.1%BSA and containing [³H]R1881 (0.01-10 nM) in the presence or absence ofa 200 fold excess of cold DHT, to determine nonspecific binding, and 1μM triamcinolone acetonide to saturate progesterone and glucocorticoidreceptors. Following a 2-hour incubation period at 37° C., cells arewashed twice with ice-cold DPBS and solubilized in DPBS containing 0.5%SDS and 20% glycerol. Extracts are removed and cell associatedradioactivity counted in a scintillation counter. The data is analyzed,including Kd and Bmax determination, by nonlinear regression usingGraphpad Prism software. When the concentration required to almostsaturate AR in both cell lines is established, the ability of the testcompounds (0.1 nM-10 μM) to displace [³H]R1881 (5.0 nM) from thereceptors is determined as described above. The IC₅₀ of each compound isdetermined by nonlinear regression with Graphpad Prism software(GraphPad Software, Inc, San Diego, Calif.).

Luciferase Transactivation Assay: Transcriptional activation assay iscarried out as described previously by Kim et al., above, with minormodifications. The Probasin luciferase reporter construct ARR2-Luc isgenerated by insertion of the minimal probasin promoter ARR2, kindlyprovided by Dr R Matusik of Vanderbilt University Medical Center(Endocrinology, 2000, 141: 4698-4710) into the polyclonal linker regionof PGL3-enhancer vector (Promega). The pRL-null (Promega) is used as theinternal control. Briefly, LNCaP cells grown in 24-well plates coatedwith poly-L-lysine were transfected with ARR2-Luc in the phenol-red freeRPMI 1640 medium containing 5% charcoal-stripped FBS (Hyclone). 24 hpost-transfection, the cells are incubated with fresh phenol-red freeserum-free RPMI 1640 medium with or without DHT and inhibitors for 18 h.Luciferase activities are measured in triplicates by using dualluciferase assay system according to the manufacturer's instruction(Promega). The results are presented as the fold induction, that is, therelative luciferase activity of the treated cells divided by that of thecontrol.

Cell Culture and Viability Assay: LNCaP cells are grown in RPMI 1640medium supplemented with 10% FBS and 1% penicillin/streptomycinsolution. To determine the effect of novel compounds on cellproliferation, cells are transferred into steroid-free medium three daysprior to the start of the experiments. Steroid-free medium consisted ofphenol red free RPMI supplemented with 5% dextran-coated, charcoaltreated serum, and 1% penicillin/streptomycin solution. Growth studiesare then performed by plating cells (3×10⁴) in 24-well multi-well dishes(Corning, Inc. Corning, N.Y.). After a 24 hours attachment period, themedium is aspirated and replaced with steroid-free medium containingvehicle or the indicated concentration of DHT (1 nM) and compounds (0.1μM-10 μM). Control wells are treated with vehicle (ethanol). This mediumis changed every three days and the number of viable cells is comparedby WST-1[4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate] assay on the seventh day. Following incubation of cells forthe above-mentioned time, 10% WST-1 solution is added to each well andincubated at 37° C. for three hours. Following incubation, plates areslightly shaken and immediately read at 450 nm with a scanningmulti-well spectrophotometer. All results represent the average of aminimum of three wells. Additional control consists of medium alone withno cells.

Pharmacokinetic Studies: All animal studies are performed according tothe guidelines and approval of the Animal Care Committee of theUniversity of Maryland School of Medicine, Baltimore. Male SCID miceweighing 20-22 gm (8-10 weeks old) obtained from NCI, Frederick, Md.,USA are maintained in a controlled environment of about 25° C., 50%relative humidity and 12 h of light and 12 h of dark cycles and allowedfree access to food and water. Compounds 5 and 6 are formulated in 40%β-cyclodextrin in water and a single subcutaneous dose is given to mice.The animals are sacrificed at various times up to 6 h after drugadministration and blood was obtained by cardiac puncture under lighthalothane (Ayerst, New York, N.Y., USA) anesthesia.

HPLC Analysis: Chromatographic separations and quantification of thesteroids and the appropriate internal standards are achieved by areverse phase HPLC method on a Waters® Novapak® C18 column (3.9×150 mm)protected by Waters® guard cartridge packed with pellicle C18 aspreviously described. Briefly, the HPLC system used in this studyconsisted of Waters® solvent delivery system, Waters® controller(Milford, Mass.), coupled to a Waters® 717^(plus) autosampler and aWaters® 996 photodiode array detector operated at 242.7 nm. The mobilephase composition is Water/MeOH/CH₃CN (35:35:30, v/v/v+200 μL of Et₃Nand 0.77 g of NH₄OAc per 1000 mL of mobile phase) at a flow rate of 1.0mL/min. The HPLC analysis is performed at ambient temperature and dataacquisition and management is achieved with a Waters® millenniumchromatography manager.

Sample Preparation: Test tubes containing mouse plasma (200 μL), 5 or 6and VN/85-1 (internal standard, 10 μL of 100 μg/mL), are extracted withdiethyl ether (2×2 mL) using a vortex mixer for 3 minutes andcentrifuged at 3000 g for 5 min. The organic layers are evaporated todryness under a gentle stream of air. The residue is reconstituted in analiquot of the mobile phase (100 μL) and filtered using 0.2 μm Teflonfilters before HPLC analysis. Calibration Curve and HPLC AssayValidation: The calibration curves for 5 in plasma and tissue and for 6in plasma are constructed by spiking varying amounts of the compoundsinto extraction tubes (duplicate) containing plasma (200 μL) and tissuepreparations (200 μL) from untreated animals to give finalconcentrations of 0.1-100.0 μg/mL. Appropriate blank extraction tubesare also prepared and an aliquot of the internal standard is added intoeach extraction tube to give a final concentration of 5 μg/ml. Thecalibration samples are taken through the sample preparation procedureas described above. An aliquot of the reconstituted extract (50 μl) isinjected into the HPLC system and the ratio of the peak areas for eachanalyte to that of the internal standard are plotted againstconcentrations of 5 or 6. The precision and accuracy of the assays aredetermined from a range of known concentrations of the inhibitors inblank plasma and taken through the HPLC procedure. The study is repeatedon three separate occasions.

Data Analysis: Pharmacokinetic calculations are performed as previouslydescribed. The non-compartmental pharmacokinetic calculations areperformed using WinNOnlin (Scientific Consulting Inc.). One-way analysisof variance (ANOVA) on SigmaStat for Windows version 1.0 is used tocompare different treatment groups at the 95% confidence level. TheBonferroni post-hoc test is used for determination of significance. AP-value of less than 0.05 is considered as statistically significant.

In Vivo Antitumor Studies (LAPC-4 Prostate Cancer Xenografts): Allanimal studies are performed according to the guidelines and approval ofthe Animal Care Committee of the University of Maryland School ofMedicine, Baltimore. Male severe combined immunodeficient (SCID) mice4-6 weeks of age purchased from the National Cancer Institute-FrederickCancer Research and Development Center (Fredrick, Md.) are housed in apathogen-free environment under controlled conditions of light andhumidity and allowed free access to food and water. Tumors are developedfrom LAPC4 cells inoculated subcutaneously (s.c.) in the miceessentially as previously described (21). LAPC4 cells are grown in IMEMwith 15% FBS plus 1% PS and 10 nm DHT until 80% confluent. Cells arescraped into DPBS, collected by centrifugation, and resuspended inMatrigel (10 mg/ml) at 3×10⁷ cells/ml. Mice are injected s.c. with 100μl of the cell suspension at one site on each flank. Tumors are measuredweekly with calipers, and tumor volumes are calculated by the formula:4/3π×r₁ ²×r₂ (r₁<r₂).

In the first experiment, LAPC4 tumors are allowed to grow for 8-10 weeksfollowing inoculation. Groups of 5 mice with comparable total tumorvolumes are either castrated or treated with 5 and 6 (0.15 mmol/kgonce-daily and 0.15 mmol/kg twice-daily, 9 a.m. and 5 p.m.). Mice arecastrated under methoxyfluorane anesthesia. Compounds 5 and 6 wereprepared at 17.2 mg/ml in a 0.3% solution of hydroxypropyl cellulose insaline, and mice receiv s.c. injections daily. Control and castratedmice are treated with vehicle only. Tumors are measured weekly for the 4weeks of treatment and tumor volumes are calculated. At the end of thetreatment period, the animals are sacrificed under halothane anesthesia;tumors are excised, weighed and stored at −80° C. Animals are alsoweighed weekly and monitored for general health status and signs ofpossible toxicity due to treatment.

In the second experiment, mice are inoculated with LAPC4 cells and aredivided into four groups of 5 mice each. The control and castrated groupreceiv vehicle, while the other two groups receiv either VN/85-1 (0.15mmol/kg twice-daily, 9 a.m. and 5 p.m.) or 5 (0.15 mmol/kg twice-daily,9 a.m. and 5 p.m.). These treatments are initiated one day after LAPC4cell inoculation; continued for 14 weeks for control group, 19 weeks(for VN/85-1 and castration groups) and for 21 weeks for 5 treated groupand tumors are measured and processed as described above.

Measurement of 5 (VN/124-1) levels in tumor, liver and testes: Theanimals in the VN/124-1-treated group are sacrificed 1 h after the lastVN/124-1 administration, and tumor, liver and testis are harvested andsnap frozen in liquid nitrogen. Tissue samples are homogenized inphosphate buffer (pH=7.4, 0.5 ml/mg of tissue). Homogenized tissue (200μl) is spiked with the internal standard, VN/85-1 (10 μL from 100 μg/mLstock solution), and then extracted with Et₂O (2×2 mL) by vortexing for3 min followed by centrifugation at 3000 g for 5 min. The Et₂O extractis separated and evaporated to dryness under a gentle stream of air. Theresidue is reconstituted in 100 μL of the HPLC mobile phase, filteredthrough 0.2 μm Teflon filters and then analyzed by HPLC as describedabove.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

TABLE 1 CYP17 and 5α-reductase activities and androgen receptor bindingof novel 17-heteroaryl compounds. 5α-Reductase % inhibition at 10 μM ARBinding CYP17 [IC₅₀ (nM)]^(b) IC₅₀ (nM)^(c) Compound^(a) IC₅₀ (nM)^(b)type 1^(d) type 2^(e) LNCaP PC3-AR  5 300.0   4 53 845 384  6 915.0 [770] [480]  1200 242  9 1250.0 ni^(f) 17 — — 10 5817.4   21 56 — — 143810.0 — — — 366 15 500.0 — — — 374 For comparison VN/85-1 50.0 — — — —Abiraterone 800.0 — — — — Ketoconazole 1100.0 — — — — Finasteride —  [60.0]   [2.0] — — Casodex — — — 940 — Flutamide — — — 11600 10985^(a)We have previously reported the synthesis of VN/85-1 (Njar et al.,above) Abiraterone was synthesized as described by Potter et al (Aconvenient, large-scale synthesis of abiraterone acetate[3β-acetoxy-17(3-pyridyl)androsta-5,16-diene], a potential new drug forthe treatment of prostate cancer. Org. Prep. Proc. Int., 1997, 29,123-128). ^(b)IC₅₀ is the concentration of inhibitor required to inhibitthe enzyme activity by 50%, each in duplicate for CYP17, triplicate for5α-reductase and AR binding. ^(c)IC₅₀ is the concentration of compoundrequired for a 50% displacement of [³H]R1881 from the androgen receptor.^(d)Prostatic tumor cell line (DU-145) expressing type 1 enzyme;substrate: 5 nM [1β-³H]androstenedione. ^(e)Enzyme from BPH tissue (type2 enzyme), 125 μg of protein, substrate: 210 nM [1β,2β-³H]testosterone.^(f)ni = no inhibition up to 10 μM. — = not determined.

TABLE 2 Pharmacokinetic parameters for 5 (50 and 100 mg/kg) and 6 (50mg/kg) after s.c. administration. 5 6 Parameter ^(a) 50 mg/kg 100 mg/kg50 mg/kg t_(1/2) (min) 44.17 ± 1.15 36.6 ± 1.6 37.93 ± 1.15 K_(el)(min⁻¹)  56.5 ± 0.94 68.49 ± 1.26 0.0183 ± 0.004 AUC (min · μg/mL)1440.00 ± 60.23  1813.94 ± 10.94  647.10 ± 20.23 T_(max) (min) 30.00 ±0.0  30.00 ± 0.0  60.00 ± 0.00 C_(max) (μg/mL) 16.82 ± 0.37 32.23 ± 0.34 5.15 ± 0.09 MRT (min) 65.40 ± 0.60 60.46 ± 1.54 79.95 ± 0.01 V_(d)(mL/kg) 2098.99 ± 4.   113276.39 ± 26.71   4207.24 ± 6.25  ^(a) Valuesare expressed as mean ± S.E., n = 5.

BRIEF DESCRIPTION OF SCHEMES AND FIGURES

Chart 1: Structures of abirateron and VN/85-1 (16)

Scheme 1: Synthesis of 17-benzoazole compounds (5, 6, 9 and 10).

Scheme 2: Synthesis of 17-diazine compounds (14 and 15).

Scheme 3: Synthesis of metabolites of trans-androsterone, includingVNLG/81.

FIG. 1: The effects of 5, 6 and casodex on transcriptional activity ofluciferase mediated through LNCaP-AR in LNCaP-ARR2-1u prostate cancercells. Cells in steroid-free medium were treated with vehicle, orincreasing concentrations of either 5 or casodex with and without 1 nMDHT for 18 h. Cells were then assayed for luciferase activity asdescribed in “Materials and Methods”. The bars represent the mean lightunits [counts per second (cps)/unit protein, i.e., relative luciferaseactivity] in triplicate wells from three separate experiments.

FIG. 2: The effects of 5, 6 and casodex on (a) LNCaP and (b) LAPC4prostate cancer cell growth. Cells were grown in steroid-free mediumbefore plating. Triplicate wells were then co-treated with increasingconcentrations of 5, 6 or casodex and DHT as described in “Materials andMethods.” The percentage (compared to control) of growth inhibitionafter 7 days of treatment was determined using WST-1 assay. The resultsrepresent the average and standard deviation of three experimentsperformed in triplicate.

FIG. 3: Typical HPLC chromatogram of 5, 16 (internal standard) andmetabolite extracted from mouse plasma. The retention times for 16,metabolite, and 5 were 11.5, 17.3 and 21.6 min, respectively.

FIG. 4: Pharmacokinetic profiles of 5 and 6 following administration ofa single subcutaneous bolus dose to male SCID mice. Each data pointrepresents the mean plasma concentions obtained from three mice. Thestandard deviations (not shown) were ±5-8% of the mean values.

FIG. 5: Pharmacokinetic profiles of 5 and metabolite following a singlesubcutaneous bolus dose (100 mg/kg.bw) of 5 to male mice.

FIG. 6: In vivo antitumor activity of 5, 6 and orchidectomy on thegrowth of LAPC4 prostate tumors in male SCID mice. Groups of five micewith LAPC-4 tumors were treated with 5 (0.15 mmol/kg/day or 0.30mmol/kg/day. Tumors volumes were measured weekly, and the percentage ofchange in tumor volume was determined after 28 days of treatment. Thestandard deviations of tumor volumes (not shown) were ±10-12% of themean values.

FIG. 7: The effects of 5, 16, and orchidectomy on the formation andgrowth of LAPC4 prostate tumors in male SCID mice. 3×10⁷ LAPC-4 cellswere injected s.c. into the dorsal flank of SCID mice. One group of micewas castrated. The other groups of mice received either vehicle or 5(0.15 mmol/kg twice-daily) or 16 (0.15 mmol/kg twice-daily). Dailytreatment with 5 or 16 was initiated 1 day after cell inoculation.Tumors volumes were measured weekly, and the percentage of change intumor volume was determined after 16 weeks of treatment. * Indicatessignificant difference of 5 versus control, castration and 16 at week 14(P=0.00065, 0.05 and 0.0097, respectively). ** Indicates significantdifference of 5 versus castration and 16 at week 16 (P=0.047 and 0.0047,respectively). ↓-↓↓: Period of reduced administered dose of 5.

1. A metabolite of a compound of the formula:


2. The metabolite of claim 1, wherein the metabolite comprises one ormore biological properties that are about the same as an analogousbiological property of a compound of the formula:


3. The metabolite of claim 2, wherein the biological property comprisesinhibition of CYP17 enzyme, inhibition of tumor growth, inhibition ofLNCaP prostate cancer cells, inhibition of LAPC4 prostate cancer cells,or androgen receptor antagonism.